Circulating leukocytes in whole blood have the potential to be used as indicators of infection, cancer, inflammation, and genetic and metabolic disease. For example, expression profiling assays have been used to identify changes in mRNA patterns that are associated with inflammation and metastatic disease and exposure to toxic and infectious agents. However, the purification of nucleic acid material from whole blood for diagnostic assays has been difficult for several reasons including the low concentration of actively metabolizing cells (only about 0.1 to 0.2% leukocytes), the high concentration of intracellular and extracellular ribonucleases, and the presence of high numbers of immature red blood cell reticulocytes which contain high levels of mRNA for α and β globin. Hemoglobin mRNA associated with immature red blood cells can lead to cross hybridization and a subsequent decrease in sensitivity and specificity during microarray experiments.
Analysis of RNA isolated from whole blood for expression patterns indicative of disease requires that the isolated RNA and (optionally) amplified RNA accurately represent the status of WBC in the whole blood sample. In this regard, transcriptional inactivation is important to achieve at the time of blood sample collection in order to obtain nucleic acid material that is intact and accurately reflects the state of the subject. It is known that mRNA is unstable in untreated whole blood samples over time. For example, adherence of monocytes to the plastic walls of tubes used for blood collection has been shown to induce mRNAs for proinflammatory cytokines (see Haskill et al., J. Immunol. 140:1690-1694 (1988)). Lag times of greater than one hour between sample collection and processing have been reported to cause alterations in several cytokine mRNAs in human whole blood (Pahl et al., Clinical Chem. 48: 2251-2253 (2002)). A recent study by Rainen et al. demonstrated an increase in some mRNA levels (e.g., interleukin 8, c-jun) and a decrease in other mRNA levels (e.g., caspase 1, heat shock protein 70) over time (4 hours, 8 hours, 3 days and 5 days) in a panel of 25 mRNAs analyzed from samples of untreated whole blood isolated from a single donor (Rainen et al., Clinical Chem. 48:1883-1890 (2002)).
Current methods for nucleic acid purification from whole blood (i.e., erythrocyte lysis and Ficoll gradient separation, etc.) are time consuming, labor intensive, and not amenable to high throughput applications. Techniques that are amenable to high throughput applications such as reverse transcription, amplification, sequencing, and microarray hybridization, require nucleic acid material that is substantially free of contaminants capable of interfering with such processing or analytical procedures. Such contaminants include substances that block or inhibit chemical reactions (e.g., nucleic acid or protein hybridizations, enzymatically catalyzed reactions, and other types of reactions used in molecular biological techniques), and substances that catalyze the degradation or de-polymerization of a nucleic acid. Contaminants also include macromolecular substances from the sample from which a nucleic acid material of interest is isolated, including enzymes, other types of proteins, red blood cells, polysaccharides, and lipids. Contaminants may also be introduced into a target nucleic acid sample from chemicals or other materials used to isolate the nucleic acid material from other substances, such as trace metals and organic solvents. Specifically with respect to the isolation of RNA from whole blood, it is important that the isolated RNA be substantially free from contamination with heme, a well known inhibitor of reverse transcription and DNA polymerase, as well as globin message, which can interfere with hybridization-based assays.
Therefore, there is a need in diagnostic molecular profiling and biomarker discovery for methods and devices that rapidly purify nucleic acid material from whole blood. In particular there is a need for methods and devices for isolating RNA from a sample of whole blood, whereby the RNA is substantially free of contaminants, including proteins, lipids, genomic DNA, globin message, and any chemicals likely to inhibit or interfere with processing or analysis of the isolated RNA, such that the isolated RNA may be subsequently analyzed using molecular biology applications that are known to be sensitive to contaminants, such as reverse transcriptase polymerase chain reaction (RT-PCR) and microarray analysis.